Deep sequencing uncovers numerous small RNAs on all four replicons of the plant pathogen Agrobacterium tumefaciens

Agrobacterium species are capable of interkingdom gene transfer between bacteria and plants. The genome of Agrobacterium tumefaciens consists of a circular and a linear chromosome, the At-plasmid and the Ti-plasmid, which harbors bacterial virulence genes required for tumor formation in plants. Little is known about promoter sequences and the small RNA (sRNA) repertoire of this and other α-proteobacteria. We used a differential RNA sequencing (dRNA-seq) approach to map transcriptional start sites of 388 annotated genes and operons. In addition, a total number of 228 sRNAs was revealed from all four Agrobacterium replicons. Twenty-two of these were confirmed by independent RNA gel blot analysis and several sRNAs were differentially expressed in response to growth media, growth phase, temperature or pH. One sRNA from the Ti-plasmid was massively induced under virulence conditions. The presence of 76 cis-antisense sRNAs, two of them on the reverse strand of virulence genes, suggests considerable antisense transcription in Agrobacterium. The information gained from this study provides a valuable reservoir for an in-depth understanding of sRNA-mediated regulation of the complex physiology and infection process of Agrobacterium

Profound Impact of Hfq on Nutrient Acquisition, Metabolism and Motility in the Plant Pathogen Agrobacterium tumefaciens

As matchmaker between mRNA and sRNA interactions, the RNA chaperone Hfq plays a key role in riboregulation of many bacteria. Often, the global influence of Hfq on the transcriptome is reflected by substantially altered proteomes and pleiotropic phenotypes in hfq mutants. Using quantitative proteomics and co-immunoprecipitation combined with RNA-sequencing (RIP-seq) of Hfq-bound RNAs, we demonstrate the pervasive role of Hfq in nutrient acquisition, metabolism and motility of the plant pathogen Agrobacterium tumefaciens. 136 of 2544 proteins identified by iTRAQ (isobaric tags for relative and absolute quantitation) were affected in the absence of Hfq. Most of them were associated with ABC transporters, general metabolism and motility. RIP-seq of chromosomally encoded Hfq 3xFlag revealed 1697 mRNAs and 209 non-coding RNAs (ncRNAs) associated with Hfq. 56 ncRNAs were previously undescribed. Interestingly, 55% of the Hfq-bound ncRNAs were encoded antisense (as) to a protein-coding sequence suggesting that A. tumefaciens Hfq plays an important role in asRNA-target interactions. The exclusive enrichment of 296 mRNAs and 31 ncRNAs under virulence conditions further indicates a role for post-transcriptional regulation in A. tumefaciens-mediated plant infection. On the basis of the iTRAQ and RIP-seq data, we assembled a comprehensive model of the Hfq core regulon in A. tumefaciens

Characterization of small RNAs in Agrobacterium tumefaciens\textit {Agrobacterium tumefaciens}

$\textit {Agrobacterium tumefaciens}$ ist ein weit verbreitetes Pflanzen-pathogenes Bakterium. In den letzten Jahren wurde die generelle Bedeutung von kleinen regulatorischen RNAs (sRNAs) in der Regulation der Genexpression in Bakterien erkannt. Ein Teil dieser Arbeit befasste sich damit kleine regulatorische RNAs (sRNAs) in $\it Agrobacterium$ zu identifizieren. RNA-Sequenzierungen (dRNA-seq) lieferten zahlreiche neue sRNAs. Ein zweites Projekt dieser Arbeit umfasste die funktionelle Charakterisierung von AbcR1, eine sRNA die eine Vielzahl an mRNAs beeinflusst. Einige dieser mRNAs kodieren für Proteine, die an der Aufnahme von Aminosäuren und Zuckern, in der Signaltransduktion der Virulenz-Kaskade und an der Nährstoffaufnahme nach Infektion von Pflanzen beteiligt sind. Die vorliegende Arbeit liefert wertvolle neue Informationen zur umfassenden sRNA- Regulation in der Physiologie von $\textit {A. tumefaciens}$ und von verwandten Bakterien, die zu Bakterien-Wirtsinteraktion befähigt sind

mRNAs enriched by Hfq^3xFlag cluster into physiological relevant groups.

<p>Clustering of Hfq^3xFlag enriched mRNAs into seven physiological coherent groups by KEGG ontology. Main groups are subdivided to specify the biological function of the respective proteins. Statistical relevance: * p-value <0.05, ** p-value <0.01.</p

Construction of <i>hfq</i>^3xFlag for co-immunoprecipitation.

<p><b>A)</b> Chromosomal integration of a <i>3xFlag</i> sequence at the 3′ end of the <i>hfq</i> sequence. <b>B)</b> Growth experiments in complex medium confirmed functionality of the Hfq^3xFlag fusion. Growth of <i>hfq</i>^WT and <i>hfq</i>^3xFlag strains was comparable, while the Δ<i>hfq</i> mutant exhibited a severe growth defect. <b>C)</b> Western blot analysis of Hfq^3xFlag protein (∼11.9 kDa) from protein extracts isolated from <i>hfq</i>^WT and <i>hfq</i>^3xFlag strains. Proteins were isolated from exponential (OD 0.5) to stationary growth phase (OD 1.5). VirB9 was detected to confirm successful induction of virulence by addition of acetosyringone (+Vir). <b>D)</b> Purification of Hfq^3xFlag and isolation of co-purified RNA. Total RNA and co-immunoprecipitated RNA (coIP RNA) were analyzed by PAA gel electrophoresis and subsequent Northern blotting. AbcR1 was detected by a DIG-labeled RNA probe. Ethidium bromide stained tRNAs served as loading control. Equal amounts of coIP RNA were loaded, but no tRNAs were detectable in the corresponding lanes.</p

Identification of Hfq-dependent ncRNAs.

<p><b>A)</b> Numbers of asRNAs and trans sRNAs enriched by Hfq^3xFlag. 152 known and 56 new ncRNAs were enriched. <b>B)</b> Expression of ncRNAs during the different growth phases from the four <i>A. tumefaciens</i> replicons. Relative expression from chromosomes (circular, linear) and mega-plasmids (At, Ti) at the different conditions is indicated in %. Northern blots of RNA isolated from WT and Δ<i>hfq</i> strains validated Hfq-dependency of trans encoded sRNA <b>C)</b> C7 and the newly identified sRNAs <b>D)</b> AhaR_C_15 and <b>E)</b> AhaR_C_17 (lower panel). Expression locus and reads from the genome browser are indicated on top. Ethidium bromide stained tRNAs served as loading control. Exp, exponential; Stat, stationary; -Vir, non-induced; +Vir, virulence-induced.</p

Hfq regulon in <i>A. tumefaciens.</i>

<p>Summary of mRNAs/proteins influenced by Hfq in <i>A. tumefaciens</i>. Results from iTRAQ and RIP-seq (this study) and AbcR1 targets <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110427#pone.0110427-Wilms3" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110427#pone.0110427-Overlper1" target="_blank">[38]</a> were combined. Operons encoding at least 2 equally regulated proteins (iTRAQ) were included and all encoded proteins were assumed to be Hfq-dependent. 21 target mRNAs simultaneously enriched with an asRNA were also included. Hfq-dependent proteins/mRNAs used for network predictions are underlined (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110427#pone-0110427-g008" target="_blank">Fig. 8</a>).</p

Hfq binds asRNAs and their cognate target mRNAs.

<p><b>A)</b> 21 of the 115 asRNAs were enriched simultaneously with their target mRNA encoded on the complementary strand. <b>B)</b> Complementarity of the 21 asRNA-mRNA pairs. 16 asRNAs were fully complementary to their designated target mRNAs. <b>C)</b>, <b>D)</b> Northern blot analysis of asRNAs and target mRNAs with full complementarity. Location and mapped reads of <b>C)</b> C1 and <i>atu0105</i> and <b>D)</b> AhaR_C_26 and <i>atu8023,</i> are indicated by the genome browser view (left). Northern blot analysis of RNA isolated from WT and Δ<i>hfq</i> strains grown under different conditions (right). Ethidium bromide stained tRNAs or 16S rRNAs served as loading control. Exp, exponential; Stat, stationary; -Vir, non-induced; +Vir, virulence-induced.</p

Quantitative proteomics of <i>A. tumefaciens</i> WT and Δ<i>hfq</i> mutant.

<p><b>A)</b> iTRAQ experiments of 3 biological replicates from stationary phase cultures of WT and Δ<i>hfq</i> mutant revealed 136 proteins differentially expressed (2544 proteins identified). <b>B)</b> Distribution of all 2544 Δ<i>hfq</i>/WT logarithmic (log) fold-changes (FC). Calculating a confidence interval of 95% resulted in an upper bound of 2.3 and a lower bound of 0.45. 100 proteins were up-regulated (FC>2.3) and 38 down-regulated (FC<0.45) in absence of <i>hfq</i>. <b>C)</b> Classification of proteins into physiological relevant groups by KEGG ontology. Filled bars indicate up- or down-regulation of proteins within the different groups. eq., equilibrium.</p

Total number of uniquely aligned reads.

<p>Total number of uniquely aligned reads.</p